GATK RealignerTargetCreator: IllegalArgumentException: Dictionary cannot have size zero

Question

[Pac314]

I am new to variant calling and trying to create realignment targets using GATK but keep getting this error, despite having a dictionary file:

 java.lang.IllegalArgumentException: Dictionary cannot have size zero
    at org.broadinstitute.gatk.utils.MRUCachingSAMSequenceDictionary.<init>(MRUCachingSAMSequenceDictionary.java:62)
    at org.broadinstitute.gatk.utils.GenomeLocParser$1.initialValue(GenomeLocParser.java:78)
    at org.broadinstitute.gatk.utils.GenomeLocParser$1.initialValue(GenomeLocParser.java:75)
    at java.lang.ThreadLocal.setInitialValue(ThreadLocal.java:180)
    at java.lang.ThreadLocal.get(ThreadLocal.java:170)
    at org.broadinstitute.gatk.utils.GenomeLocParser.getContigInfo(GenomeLocParser.java:91)
    at org.broadinstitute.gatk.utils.GenomeLocParser.getContigs(GenomeLocParser.java:204)
    at org.broadinstitute.gatk.utils.GenomeLocParser.<init>(GenomeLocParser.java:135)
    at org.broadinstitute.gatk.utils.GenomeLocParser.<init>(GenomeLocParser.java:108)
    at org.broadinstitute.gatk.utils.GenomeLocSortedSet.createSetFromSequenceDictionary(GenomeLocSortedSet.java:421)
    at org.broadinstitute.gatk.engine.datasources.reads.BAMScheduler.createOverMappedReads(BAMScheduler.java:66)
    at org.broadinstitute.gatk.engine.datasources.reads.IntervalSharder.shardOverMappedReads(IntervalSharder.java:55)
    at org.broadinstitute.gatk.engine.datasources.reads.SAMDataSource.createShardIteratorOverMappedReads(SAMDataSource.java:1217)
    at org.broadinstitute.gatk.engine.GenomeAnalysisEngine.getShardStrategy(GenomeAnalysisEngine.java:649)
    at org.broadinstitute.gatk.engine.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:307)
    at org.broadinstitute.gatk.engine.CommandLineExecutable.execute(CommandLineExecutable.java:113)
    at org.broadinstitute.gatk.utils.commandline.CommandLineProgram.start(CommandLineProgram.java:255)
    at org.broadinstitute.gatk.utils.commandline.CommandLineProgram.start(CommandLineProgram.java:157)
    at org.broadinstitute.gatk.engine.CommandLineGATK.main(CommandLineGATK.java:108)

My code for the command is:

java -jar -Xmx8G gatk.jar \
   -T RealignerTargetCreator \
   -R $ref \
   -I sample_marked.bam \
   -o sample_realignment_targets.list

The dictionary contains this:

@HD VN:1.5
@SQ SN:ST4  LN:61165649 M5:d17409fc1c12bcd44fcb59e2803c3659 UR:file:~/varcalling/reference.fa

Please could you help me solve this issue.

[Pierre Lindenbaum]

what are the outputs of

wc -l ref.fa.fai ref.dict
file  ref.fa.fai ref.dict
samtools view -H sample_marked.bam | grep '@SQ'

[Pac314]

Thanks for you reply.

For this wc -l ref.fa.fai ref.dict the output is:

1 ref.fa.fai
2 ref.fa.dict
3 total

For file ref.fa.fai ref.dict:

ref.fa.fai:  ASCII text
ref.dict: ASCII text

For samtools view -H sample_marked.bam | grep '@SQ': There is not any output.

[Pierre Lindenbaum]

There is not any output.

this is your problem. The BAM file is missing a dictionary. Check your upstream workflow.

[Pac314]

After I have marked the duplicates in the BAM, the BAM has an "@SQ" line but lacks read groups. When I manually add reads groups using AddOrReplaceReadGroups the BAM loses the "@SQ" line but retains the read groups. Why would this happen?